Atrial fibrillation is a growing public health problem without adequate therapies. Angiotensin II (Ang II) and reactive oxygen species (ROS) are validated risk factors for atrial fibrillation (AF) in patients, but the molecular pathway(s) connecting ROS and AF is unknown. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a ROS activated proarrhythmic signal, so we hypothesized that oxidized CaMKIIδ(ox-CaMKII) could contribute to AF.
Methods and Results
We found ox-CaMKII was increased in atria from AF patients compared to patients in sinus rhythm and from mice infused with Ang II compared with saline. Ang II treated mice had increased susceptibility to AF compared to saline treated WT mice, establishing Ang II as a risk factor for AF in mice. Knock in mice lacking critical oxidation sites in CaMKIIδ (MM-VV) and mice with myocardial-restricted transgenic over-expression of methionine sulfoxide reductase A (MsrA TG), an enzyme that reduces ox-CaMKII, were resistant to AF induction after Ang II infusion.
Our studies suggest that CaMKII is a molecular signal that couples increased ROS with AF and that therapeutic strategies to decrease ox-CaMKII may prevent or reduce AF.
atrial fibrillation; arrhythmia mechanisms; calcium/calmodulin-dependent protein kinase II; angiotensin II; reactive oxygen species
CaV1.2; phosphorylation; protein kinases
Increased reactive oxygen species (ROS) contribute to asthma, but little is known about the molecular mechanisms connecting increased ROS with characteristic features of asthma. We show that enhanced oxidative activation of the Ca2+/calmodulin-dependent protein kinase (ox-CaMKII) in bronchial epithelium positively correlates with asthma severity and that epithelial ox-CaMKII increases in response to inhaled allergens in patients. We used mouse models of allergic airway disease induced by ovalbumin (OVA) or Aspergillus fumigatus (Asp) and found that bronchial epithelial ox-CaMKII was required to increase a ROS- and picrotoxin-sensitive Cl− current (ICl) and MUC5AC expression, upstream events in asthma progression. Allergen challenge increased epithelial ROS by activating NADPH oxidases. Mice lacking functional NADPH oxidases due to knockout of p47 and mice with epithelial-targeted transgenic expression of a CaMKII inhibitory peptide or wild-type mice treated with inhaled KN-93, an experimental small molecule CaMKII antagonist, were protected against increases in ICl, MUC5AC expression, and airway hyper-reactivity to inhaled methacholine. Our findings support the view that CaMKII is a ROS-responsive, pluripotent pro-asthmatic signal and provide proof-of-concept evidence that CaMKII is a therapeutic target in asthma.
Ca2+ plays a crucial role in connecting membrane excitability with contraction in myocardium. The hallmark features of heart failure are mechanical dysfunction and arrhythmias; defective intracellular Ca2+ homeostasis is a central cause of contractile dysfunction and arrhythmias in failing myocardium. Defective Ca2+ homeostasis in heart failure can result from pathological alteration in the expression and activity of an increasingly understood collection of Ca2+ homeostatic binding proteins, ion channels and enzymes. This review focuses on the molecular mechanisms of defective Ca2+ cycling in heart failure and consider how fundamental understanding of these pathways may translate into novel and innovative therapies.
Calcium; heart failure; excitation-contraction coupling; CaMKII; mitochondria
The calcium and calmodulin-dependent protein kinase II (CaMKII) is present in sinoatrial node (SAN) pacemaker cells and is required for physiological “fight or flight” SAN beating rate responses. Inhibition of CaMKII in SAN does not affect baseline heart rate, but reduces heart rate increases in response to physiological stress. CaMKII senses intracellular calcium (Ca2+) changes, oxidation status, and hyperglycemia to phosphorylate substrates that regulate Ca2+-sensitive proteins, such as L-type Ca2+ channels, phospholamban, and cardiac ryanodine receptors (RyR2). All of these substrates are involved in the SAN pacemaking mechanism. Excessive CaMKII activity, as occurs under pathological conditions such as heart failure, ischemia, and diabetes, can promote intracellular Ca2+ overload and reactive oxygen species production. Oxidation of CaMKII (ox-CaMKII) locks CaMKII into a constitutively active configuration that contributes to SAN cell apoptosis and fibrosis. This ox-CaMKII-mediated loss of functional SAN cells contributes to SAN dysfunction (SND) and sudden death. Thus, CaMKII has emerged as a central regulator of physiological SAN responses and a key determinant of SND.
calcium/calmodulin-dependent protein kinase II; sinoatrial node; heart rate; sinoatrial node dysfunction; calcium
The sodium-calcium exchanger 1 (NCX1) is predominantly expressed in the heart and is implicated in controlling automaticity in isolated sinoatrial nodal (SAN) pacemaker cells, but the potential role of NCX1 in determining heart rate in vivo is unknown.
Determine the role of Ncx1 in heart rate.
Methods and Results
We employed global myocardial and SAN-targeted conditional Ncx1 knockout (Ncx1−/−) mice to measure the effect of the NCX current (INCX) in pacemaking activity in vivo, ex vivo and in isolated SAN cells. We induced conditional Ncx1−/− using a Cre/loxP system. Unexpectedly, in vivo and ex vivo hearts and isolated SAN cells showed that basal rates in Ncx1−/− (retaining ~20% of control level INCX) and control mice were similar, suggesting that physiological NCX1 expression is not required for determining resting heart rate. However, heart rate and SAN cell automaticity increases in response to isoproterenol or the dihydropyridine Ca2+ channel agonist BayK8644 were significantly blunted or eliminated in Ncx1−/− mice, indicating that NCX1 is important for fight or flight heart rate responses. In contrast the ‘pacemaker’ current (If) and L-type Ca2+ currents were equivalent in control and Ncx1−/− SAN cells under resting and isoproterenol-stimulated conditions. Ivabradine, an If antagonist with clinical efficacy, reduced basal SAN cell automaticity similarly in control and Ncx1−/− mice. However, ivabradine decreased automaticity in SAN cells isolated from Ncx1−/− mice more effectively than in control SAN cells after isoproterenol, suggesting that the importance of INCX in fight or flight rate increases is enhanced after If inhibition.
Physiological Ncx1 expression is required for increasing sinus rates in vivo, ex vivo and in isolated SAN cells but not for maintaining resting heart rate.
Na+-Ca2+ exchange; sinoatrial node; L-type Ca2+ channels; pacemaker current; ion channel
arrhythmia (mechanisms); calcium; ion channels or ion channel; structural heart disease; CaMKII
Understanding relationships between heart failure and arrhythmias, important causes of suffering and sudden death, remains an unmet goal for biomedical researchers and physicians. Evidence assembled over the last decade supports a view that activation of the multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) favors myocardial dysfunction and cell membrane electrical instability. CaMKII activation follows increases in intracellular Ca2+ or oxidation, upstream signals with the capacity to transition CaMKII into a Ca2+ and calmodulin-independeant, constitutively active enzyme. Constitutively active CaMKII appears poised to participate in disease pathways by catalyzing the phosphorylation of classes of protein targets important for excitation-contraction coupling and cell survival, including ion channels and Ca2+ homeostatic proteins, and transcription factors that drive hypertrophic and inflammatory gene expression. This rich diversity of downstream targets helps to explain the potential for CaMKII to simultaneously affect mechanical and electrical properties of heart muscle cells. Proof of concept studies from a growing number of investigators show that CaMKII inhibition is beneficial for improving myocardial performance and reducing arrhythmias. Here we review the molecular physiology of CaMKII, discuss CaMKII actions at key cellular targets and results of animal models of myocardial hypertrophy, dysfunction and arrhythmias that suggest CaMKII inhibition may benefit myocardial function while reducing arrhythmias.
CaMKII; Arrhythmias; Heart Failure; Ion channels; Remodeling
Calcium uptake through the mitochondrial Ca2+ uniporter (MCU) is thought to be essential in regulating cellular signaling events, energy status, and survival. Functional dissection of the uniporter is now possible through the recent identification of the genes encoding for MCU protein complex subunits. Cancer cells exhibit many aspects of mitochondrial dysfunction associated with altered mitochondrial Ca2+ levels including resistance to apoptosis, increased reactive oxygen species production and decreased oxidative metabolism. We used a publically available database to determine that breast cancer patient outcomes negatively correlated with increased MCU Ca2+ conducting pore subunit expression and decreased MICU1 regulatory subunit expression. We hypothesized breast cancer cells may therefore be sensitive to MCU channel manipulation. We used the widely studied MDA-MB-231 breast cancer cell line to investigate whether disruption or increased activation of mitochondrial Ca2+ uptake with specific siRNAs and adenoviral overexpression constructs would sensitize these cells to therapy-related stress. MDA-MB-231 cells were found to contain functional MCU channels that readily respond to cellular stimulation and elicit robust AMPK phosphorylation responses to nutrient withdrawal. Surprisingly, knockdown of MCU or MICU1 did not affect reactive oxygen species production or cause significant effects on clonogenic cell survival of MDA-MB-231 cells exposed to irradiation, chemotherapeutic agents, or nutrient deprivation. Overexpression of wild type or a dominant negative mutant MCU did not affect basal cloning efficiency or ceramide-induced cell killing. In contrast, non-cancerous breast epithelial HMEC cells showed reduced survival after MCU or MICU1 knockdown. These results support the conclusion that MDA-MB-231 breast cancer cells do not rely on MCU or MICU1 activity for survival in contrast to previous findings in cells derived from cervical, colon, and prostate cancers and suggest that not all carcinomas will be sensitive to therapies targeting mitochondrial Ca2+ uptake mechanisms.
Recent studies suggest that proarrhythmic effects of cardiac glycosides (CGs) on cardiomyocyte Ca2+ handling involve generation of reactive oxygen species (ROS). However, the specific pathway(s) of ROS production and the subsequent downstream molecular events that mediate CG-dependent arrhythmogenesis remain to be defined.
Methods and results
We examined the effects of digitoxin (DGT) on Ca2+ handling and ROS production in cardiomyocytes using a combination of pharmacological approaches and genetic mouse models. Myocytes isolated from mice deficient in NADPH oxidase type 2 (NOX2KO) and mice transgenically overexpressing mitochondrial superoxide dismutase displayed markedly increased tolerance to the proarrhythmic action of DGT as manifested by the inhibition of DGT-dependent ROS and spontaneous Ca2+ waves (SCW). Additionally, DGT-induced mitochondrial membrane potential depolarization was abolished in NOX2KO cells. DGT-dependent ROS was suppressed by the inhibition of PI3K, PKC, and the mitochondrial KATP channel, suggesting roles for these proteins, respectively, in activation of NOX2 and in mitochondrial ROS generation. Western blot analysis revealed increased levels of oxidized CaMKII in WT but not in NOX2KO hearts treated with DGT. The DGT-induced increase in SCW frequency was abolished in myocytes isolated from mice in which the Ser 2814 CaMKII phosphorylation site on RyR2 is constitutively inactivated.
These results suggest that the arrhythmogenic adverse effects of CGs on Ca2+ handling involve PI3K- and PKC-mediated stimulation of NOX2 and subsequent NOX2-dependent ROS release from the mitochondria; mitochondria-derived ROS then activate CaMKII with consequent phosphorylation of RyR2 at Ser 2814.
Calcium; Reactive oxygen species; NADPH oxidase; Mitochondria; CaMKII
Increasing heart rate enhances cardiac contractility (force frequency relationship, FFR) and accelerates cardiac relaxation (frequency-dependent acceleration of relaxation, FDAR). The positive FFR together with FDAR promotes rapid filling and ejection of blood from the left ventricle (LV) at higher heart rates. Recent studies indicate that the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is involved in regulating FFR and FDAR. We used isolated perfused mouse hearts to study the mechanisms of FFR and FDAR in different genetic models, including transgenic myocardial CaMKII inhibition (AC3-I) and phosphalamban knockout (PLN−/−). When the rate was increased from 360 beats/min to 630 beats/min in wild type mouse hearts, the LV developed pressure (LVDP) and the maximum rate of increase in pressure (dP/dt max) increased by 37.6 ± 4.7% and 77.0 ± 8.1%, respectively. However, hearts from AC3-I littermates showed no increase of LVDP and a relatively modest (20.4 ± 3.9 %) increase in dP/dt max. PLN−/− hearts had a negative FFR, and myocardial AC3-I expression did not change the FFR in PLN−/− mice. PLN−/− mouse hearts did not exhibit FDAR, while PLN−/−mice with myocardial AC3-I expression showed further frequency dependent reductions in cardiac relaxation, suggesting CaMKII targets in addition to PLN were critical to myocardial relaxation. We incubated a constitutively active form of CaMKII with chemically-skinned myocardium and found that several myofilament proteins were phosphorylated by CaMKII. However, CaMKII did not affect myofilament calcium sensitivity. Our study shows that CaMKII plays an important role in modulating FFR and FDAR in murine hearts and suggest that PLN is a critical target for CaMKII effects on FFR, while CaMKII effects on FDAR partially require PLN-alternative targets.
CaM kinase II; force-frequency relation; frequency-dependent acceleration of relaxation; phospholamban
Diabetes increases oxidant stress and doubles the risk of dying after myocardial
infarction, but the mechanisms underlying increased mortality are unknown. Mice with
streptozotocin-induced diabetes developed profound heart rate slowing and doubled
mortality compared with controls after myocardial infarction. Oxidized
Ca2+/calmodulin-dependent protein kinase II (ox-CaMKII) was
significantly increased in pacemaker tissues from diabetic patients compared with
that in nondiabetic patients after myocardial infarction. Streptozotocin-treated mice
had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by
myocardial infarction. We developed a knockin mouse model of oxidation-resistant
CaMKIIδ (MM-VV), the isoform associated with cardiovascular disease.
Streptozotocin-treated MM-VV mice and WT mice infused with MitoTEMPO, a mitochondrial
targeted antioxidant, expressed significantly less ox-CaMKII, exhibited increased
pacemaker cell survival, maintained normal heart rates, and were resistant to
diabetes-attributable mortality after myocardial infarction. Our findings suggest
that activation of a mitochondrial/ox-CaMKII pathway contributes to increased sudden
death in diabetic patients after myocardial infarction.
Aldosterone contributes to the endocrine basis of heart failure and studies on cardiac aldosterone signaling have reinforced its value as a therapeutic target. Recent focus has shifted to new roles of aldosterone that appear to depend on co-existing pathologic stimuli, cell type, and disease etiology. This review evaluates recent advances in mechanisms underlying aldosterone-induced cardiac disease and highlights the interplay between aldosterone and Ca2+ and calmodulin dependent protein kinase II, whose hyperactivity during heart failure contributes to disease progression. Increasing evidence implicates aldosterone in diastolic dysfunction, and there is need to develop more targeted therapeutics such as aldosterone synthase inhibitors and molecularly specific anti-oxidants. Despite accumulating knowledge, many questions still persist and will likely dictate areas of future research.
aldosterone; CaMKII; heart failure; signaling
Emerging evidence indicates a critical role for junctophilin-2 (JP2) in T-tubule integrity and assembly of cardiac dyads in adult ventricular myocytes. In the postnatal stage, one of the critical features of myocyte maturation is development of the T-tubule system, though the mechanisms remain poorly understood. In this study, we aim to determine whether JP2 is required for normal cardiac T-tubule maturation.
Methods and results
Using in situ confocal imaging of intact murine hearts, we found T-tubules were absent in both left- and right-ventricular myocytes at postnatal Day 8 and did not appear until Day 10. Quantification of T-tubule structural integrity using the T-tubule power (TTpower) index revealed a progressive increase in TTpower between postnatal Days 10 and 19. By postnatal Day 19, TTpower was similar to that in adult murine cardiomyocytes, indicative of a nearly matured T-tubule network. JP2 levels increased dramatically during development, reaching levels observed in adult hearts by postnatal Day 14. Deficiency of JP2, using a mouse model in which a JP2-specific shRNA is expressed during embryonic development, severely impaired T-tubule maturation, with equivalent decreases in the left- and right-ventricular TTpower. We also detected a gradual increase in the density of transverse but not longitudinal tubules during development, and JP2 deficiency abolished the increase in the density of transverse elements. Alterations in T-tubules caused significant reduction in Ca2+ transient amplitude and marked increase in Ca2+ release dyssynchrony, Ca2+ alternans, and spontaneous Ca2+ waves, leading to contractile failure.
Our data identify a critical role for JP2 in T-tubule and excitation–contraction coupling maturation during development.
T-tubules; Junctophilin-2; Excitation–contraction coupling; Calcium; Heart development
Catecholamines increase heart rate by augmenting the cAMP responsive HCN4 ‘pacemaker current’ (If) and/or by promoting inward Na+/Ca2+ exchanger current (INCX), by a ‘Ca2+ clock’ mechanism in sinoatrial nodal cells (SANCs). The importance, identity and function of signals that connect If and Ca2+ clock mechanisms are uncertain and controversial, but the multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) is required for physiological heart rate responses to β-adrenergic receptor (β-AR) stimulation. The aim of this stuy is to measure the contribution of the Ca2+ clock and CaMKII to cardiac pacing independent of β-AR agonist stimulation.
Methods and Results
We used the L-type Ca2+ channel agonist BayK 8644 (BayK) to activate the SANC Ca2+ clock. BayK and isoproterenol were similarly effective in increasing rates in SANCs and Langendorff-perfused hearts from WT control mice. In contrast, SANCs and isolated hearts from mice with CaMKII inhibition by transgenic expression of an inhibitory peptide (AC3-I) were resistant to rate increases by BayK. BayK only activated CaMKII in control SANCs, but increased ICa equally in all SANCs, indicating that increasing ICa was insufficient and suggesting CaMKII activation was required for heart rate increases by BayK. BayK did not increase If or protein kinase A (PKA)-dependent phosphorylation of phospholamban (at Ser16), indicating that increased SANC Ca2+ by BayK did not augment cAMP/PKA signaling at these targets. Late diastolic intracellular Ca2+ release and INCX were significantly reduced in AC3-I SANCs and the response to BayK was eliminated by ryanodine in all groups.
The Ca2+ clock is capable of supporting physiological fight or flight responses, independent of β-AR stimulation or If increases. Complete Ca2+ clock and β-AR stimulation responses require CaMKII.
Ca2+/calmodulin-dependent protein kinase (CaMKII); sinoatrial node cells; L-type Ca2+ channels; pacemaker current; sarcoplasmic reticulum
Myocardial infarction is a major cause of morbidity and mortality in the developing and developed world. Although current interventions have been successful in prolonging life, they are inadequate because mortality is still high among MI patients. The multifunctional Ca2+/calmodulin dependent protein kinase (CaMKII) plays a key role in the structure and contractility of the myocardium. CaMKII activity is increased in MI hearts and CaMKII promotes cardiac hypertrophy and inflammation, processes consistently activated by myocardial injury. Hypertrophy and inflammation are also related to neurohumoral and redox signaling that uncouple CaMKII activation from Ca2+/calmodulin dependence. Thus, CaMKII may act as a nodal point for integrating hypertrophic and inflammatory signaling in myocardium.
CaMKII; Heart; Myocardium; Myocardial infarction; Heart attack; Inflammation; Hypertrophy; Toll-like receptors; Oxidative stress; ROS; AngII
In cardiac muscle, the release of calcium ions from the sarcoplasmic reticulum through ryanodine receptor ion channels (RyR2s) leads to muscle contraction. RyR2 is negatively regulated by calmodulin (CaM) and by phosphorylation of Ca2+/CaM-dependent protein kinase II (CaMKII). Substitution of three amino acid residues in the CaM binding domain of RyR2 (RyR2-W3587A/L3591D/F3603A, RyR2ADA) impairs inhibition of RyR2 by CaM and results in cardiac hypertrophy and early death of mice carrying the RyR2ADA mutation. To test the cellular function of CaMKII in cardiac hypertrophy, mutant mice were crossed with mice expressing the CaMKII inhibitory AC3-I peptide or the control AC3-C peptide in the myocardium. Inhibition of CaMKII by AC3-I modestly reduced CaMKII-dependent phosphorylation of RyR2 at Ser-2815 and markedly reduced CaMKII-dependent phosphorylation of SERCA2a regulatory subunit phospholamban at Thr-17. However the average life span and heart-to-body weight ratio of Ryr2ADA/ADA mice expressing the inhibitory peptide were not altered compared to control mice. In Ryr2ADA/ADA homozygous mice, AC3-I did not alter cardiac morphology, enhance cardiac function, improve sarcoplasmic reticulum Ca2+ handling, or suppress the expression of genes implicated in cardiac remodeling. The results suggest that CaMKII was not required for the rapid development of cardiac hypertrophy in Ryr2ADA/ADA mice.
The multifunctional calcium/calmodulin-dependent kinase II (CaMKII) is activated by vasoconstrictors in vascular smooth muscle cells (VSMC), but its impact on vasoconstriction remains unknown. We hypothesized that CaMKII inhibition in VSMC decreases vasoconstriction. Using novel transgenic mice that express the inhibitor peptide CaMKIIN in smooth muscle (TG SM-CaMKIIN), we investigated the effect of CaMKII inhibition on L-type Ca2+ channel (LTCC) current (ICa), cytoplasmic and sarcoplasmic reticulum (SR) Ca2+ and vasoconstriction in mesenteric arteries. In mesenteric VSMC, CaMKII inhibition significantly reduced action potential duration and the residual ICa 50 ms after peak amplitude, indicative of loss of LTCC-dependent ICa facilitation. Treatment with angiotensin-II or phenylephrine increased the intracellular Ca2+ concentration ([Ca2+]i) in WT but not TG SM-CaMKIIN VSMC. The difference in [Ca2+]i was abolished by pretreatment with nifedipine, an LTCC antagonist. In TG SM-CaMKIIN VSMC, the total SR Ca2+ content was reduced as a result of diminished SR Ca2+ ATPase (SERCA) activity via impaired derepression of the SERCA inhibitor phospholamban. Despite the differences in [Ca2+]i, CaMKII inhibition did not alter myogenic tone or vasoconstriction of mesenteric arteries in response to KCl, angiotensin-II and phenylephrine. However, it increased myosin light chain kinase activity. These data suggest that CaMKII activity maintains intracellular calcium homeostasis but is not required for vasoconstriction of mesenteric arteries.
CaMKII; Ca2+ signaling; contraction; L-type Ca2+ channel
Timothy Syndrome (TS) is a disease of excessive cellular Ca2+ entry and life-threatening arrhythmias due to a mutation in the primary cardiac L-type Ca2+ channel (CaV1.2). The TS mutation causes loss of normal voltage-dependent inactivation (VDI) of CaV1.2 current (ICa). During cellular Ca2+ overload the calmodulin-dependent protein kinase II (CaMKII) causes arrhythmias. We hypothesized that CaMKII is a part of the proarrhythmic mechanism in TS.
Methods and Results
We developed an adult rat ventricular myocyte model of TS (G406R) by lenti virus-mediated transfer of wild type (WT) and TS CaV1.2. The exogenous CaV1.2 contained a mutation (T1066Y) conferring dihydropyridine resistance, so we could silence endogenous CaV1.2 with nifedipine and maintain peak ICa at control levels in infected cells. TS CaV1.2 infected ventricular myocytes exhibited the signature VDI loss under Ca2+ buffering conditions, not permissive for CaMKII activation. In physiological Ca2+ solutions, TS CaV1.2 expressing ventricular myocytes exhibited increased CaMKII activity and a proarrhythmic phenotype that included action potential prolongation, increased ICa facilitation and afterdepolarizations. Intracellular dialysis of a CaMKII inhibitory peptide, but not a control peptide, reversed increases in ICa facilitation, normalized the action potential and prevented afterdepolarizations. We developed a revised mathematical model that accounts for CaMKII-dependent and CaMKII-independent effects of the TS mutation.
In TS the loss of VDI is an upstream initiating event for arrhythmia phenotypes that are ultimately dependent on CaMKII activation.
action potentials; calcium; ion channels; myocytes
ATP-sensitive potassium (KATP) channels have the unique ability to adjust membrane excitability and functions in accordance with the metabolic status of the cell. Skeletal muscles are primary sites of activity-related energy consumption and have KATP channels expressed in very high density. Previously, we demonstrated that transgenic mice with skeletal muscle–specific disruption of KATP channel function consume more energy than wild-type littermates. However, how KATP channel activation modulates skeletal muscle resting and action potentials under physiological conditions, particularly low-intensity workloads, and how this can be translated to muscle energy expenditure are yet to be determined. Here, we developed a technique that allows evaluation of skeletal muscle excitability in situ, with minimal disruption of the physiological environment. Isometric twitching of the tibialis anterior muscle at 1 Hz was used as a model of low-intensity physical activity in mice with normal and genetically disrupted KATP channel function. This workload was sufficient to induce KATP channel opening, resulting in membrane hyperpolarization as well as reduction in action potential overshoot and duration. Loss of KATP channel function resulted in increased calcium release and aggravated activity-induced heat production. Thus, this study identifies low-intensity workload as a trigger for opening skeletal muscle KATP channels and establishes that this coupling is important for regulation of myocyte function and thermogenesis. These mechanisms may provide a foundation for novel strategies to combat metabolic derangements when energy conservation or dissipation is required.
Sinus node dysfunction (SND) is a major public health problem that is associated with sudden cardiac death and requires surgical implantation of artificial pacemakers. However, little is known about the molecular and cellular mechanisms that cause SND. Most SND occurs in the setting of heart failure and hypertension, conditions that are marked by elevated circulating angiotensin II (Ang II) and increased oxidant stress. Here, we show that oxidized calmodulin kinase II (ox-CaMKII) is a biomarker for SND in patients and dogs and a disease determinant in mice. In wild-type mice, Ang II infusion caused sinoatrial nodal (SAN) cell oxidation by activating NADPH oxidase, leading to increased ox-CaMKII, SAN cell apoptosis, and SND. p47–/– mice lacking functional NADPH oxidase and mice with myocardial or SAN-targeted CaMKII inhibition were highly resistant to SAN apoptosis and SND, suggesting that ox-CaMKII–triggered SAN cell death contributed to SND. We developed a computational model of the sinoatrial node that showed that a loss of SAN cells below a critical threshold caused SND by preventing normal impulse formation and propagation. These data provide novel molecular and mechanistic information to understand SND and suggest that targeted CaMKII inhibition may be useful for preventing SND in high-risk patients.
Increasing evidence suggests that cardiac pacemaking is the result of two sinoatrial node (SAN) cell mechanisms: a ‘voltage clock’ and a Ca2+ dependent process, or ‘Ca2+ clock.’ The voltage clock initiates action potentials (APs) by SAN cell membrane potential depolarization from inward currents, of which the pacemaker current (If) is thought to be particularly important. A Ca2+ dependent process triggers APs when sarcoplasmic reticulum (SR) Ca2+ release activates inward current carried by the forward mode of the electrogenic Na+/Ca2+ exchanger (NCX). However, these mechanisms have mostly been defined in rodents or rabbits, but are unexplored in single SAN cells from larger animals. Here, we used patch-clamp and confocal microscope techniques to explore the roles of the voltage and Ca2+ clock mechanisms in canine SAN pacemaker cells. We found that ZD7288, a selective If antagonist, significantly reduced basal automaticity and induced irregular, arrhythmia-like activity in canine SAN cells. In addition, ZD7288 impaired but did not eliminate the SAN cell rate acceleration by isoproterenol. In contrast, ryanodine significantly reduced the SAN cell acceleration by isoproterenol, while ryanodine reduction of basal automaticity was modest (∼14%) and did not reach statistical significance. Importantly, pretreatment with ryanodine eliminated SR Ca2+ release, but did not affect basal or isoproterenol-enhanced If. Taken together, these results indicate that voltage and Ca2+ dependent automaticity mechanisms coexist in canine SAN cells, and suggest If and SR Ca2+ release cooperate to determine baseline and catecholamine-dependent automaticity in isolated dog SAN cells.
sinoatrial node cells; action potentials; funny current; sarcoplasmic reticulum; pacemaker
Ventricular tachycardia (VT) is the second most common cause of death in patients with Duchenne muscular dystrophy (DMD). Recent studies have implicated enhanced sarcoplasmic reticulum (SR) Ca2+ leak via ryanodine receptors (RyR2) as a cause of VT in the mdx mouse model of DMD. However, the signaling mechanisms underlying induction of SR Ca2+ leak and VT are poorly understood.
To test whether enhanced CaMKII phosphorylation of RyR2 underlies SR Ca2+ leak and induction of VT in mdx mice.
Programmed electrical stimulation (PES) was performed on anesthetized mice, and confocal imaging of calcium release events in isolated ventricular myocytes.
PES revealed inducible VT in mdx mice, which was inhibited by CaMKII inhibition or mutation S2814A in RyR2. Myocytes from mdx mice exhibited more Ca2+ sparks and Ca2+ waves compared with wild type (WT) mice, in particular at faster pacing rates. Arrhythmogenic Ca2+ waves were inhibited by CaMKII but not PKA inhibition. Moreover, mutation S2814A but not S2808A in RyR2 suppressed spontaneous Ca2+ waves in myocytes from mdx mice.
CaMKII blockade and genetic inhibition of RyR2-S2814 phosphorylation prevent VT induction in a mouse model of DMD. In ventricular myocytes from mdx mice, spontaneous Ca2+ sparks and Ca2+ waves can be suppressed by CaMKII inhibition or mutation S2814A in RyR2. Thus, inhibition of CaMKII-induced SR Ca2+ leak might be a new strategy to prevent arrhythmias in patients with DMD without heart failure.
Cardiac arrhythmias; Ca2+/calmodulin kinase II; mouse model; ryanodine receptor; Duchenne muscular dystrophy; ventricular tachycardia
Clinical use of the widely used chemotherapeutic agent doxorubicin is limited by life-threatening cardiotoxicity. The mechanisms underlying Dox-induced cardiomyopathy and heart failure remain unclear, but are thought to involve p53-mediated myocardial cell apoptosis. The tripartite G protein inactivating protein RGS6 has been implicated in reactive oxygen species (ROS) generation, ATM/p53 activation and apoptosis in Dox-treated cells. Thus, we hypothesized that RGS6, the expression of which is enriched in cardiac tissue, might also be responsible for the pathological effects of Dox treatment in heart. In this study, we show that RGS6 expression is induced strongly by Dox in the ventricles of mice and isolated ventricular myocytes (VCM) via a post-transcriptional mechanism. While Dox-treated wild type (WT) mice manifested severe left ventricular dysfunction, loss of heart and body mass, along with decreased survival five days after Dox administration, mice lacking RGS6 were completely protected against these pathogenic responses. Activation of ATM/p53-apoptosis signaling by Dox in ventricles of WT mice was also absent in their RGS6−/− counterparts. Dox-induced ROS generation was dramatically impaired in both the ventricles and VCM isolated from RGS6−/− mice, and the apoptotic response to Dox in VCM required RGS6-dependent ROS production. These results identify RGS6 as an essential mediator of the pathogenic responses to Dox in heart, and they argue that RGS6 inhibition offers a rational means to circumvent Dox cardiotoxicity in human cancer patients.
RGS6; doxorubicin cardiotoxicity; apoptosis; p53; reactive oxygen species
Many signals have risen and fallen in the tide of investigation into mechanisms of myocardial hypertrophy and heart failure (HF). In our opinion, the multifunctional Ca and calmodulin-dependent protein kinase II (CaMKII) has emerged as a molecule to watch, in part because a solid body of accumulated data essentially satisfy Koch's postulates, showing that the CaMKII pathway is a core mechanism for promoting myocardial hypertrophy and heart failure. Multiple groups have now confirmed the following: (1) that CaMKII activity is increased in hypertrophied and failing myocardium from animal models and patients; (2) CaMKII overexpression causes myocardial hypertrophy and HF and (3) CaMKII inhibition (by drugs, inhibitory peptides and gene deletion) improves myocardial hypertrophy and HF. Patients with myocardial disease die in equal proportion from HF and arrhythmias, and a major therapeutic obstacle is that drugs designed to enhance myocardial contraction promote arrhythmias. In contrast, inhibiting the CaMKII pathway appears to reduce arrhythmias and improve myocardial responses to pathological stimuli. This brief paper will introduce the molecular physiology of CaMKII and discuss the impact of CaMKII on ion channels, Ca handling proteins and transcription in myocardium. This article is part of a Special Issue entitled “Key Signaling Molecules Special Issue”.
Calmodulin kinase II; Cell signaling; Oxidation; Hypertrophy; Heart failure; Arrhythmias